CN105169377A - Novel application of lysozyme-antibacterial peptide fusion proteins - Google Patents
Novel application of lysozyme-antibacterial peptide fusion proteins Download PDFInfo
- Publication number
- CN105169377A CN105169377A CN201510478272.7A CN201510478272A CN105169377A CN 105169377 A CN105169377 A CN 105169377A CN 201510478272 A CN201510478272 A CN 201510478272A CN 105169377 A CN105169377 A CN 105169377A
- Authority
- CN
- China
- Prior art keywords
- lysozyme
- antibacterial peptide
- peptide fusion
- fusion rotein
- wound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the field of biochemistry, and discloses novel application of lysozyme-antibacterial peptide fusion proteins. The lysozyme-antibacterial peptide fusion proteins can be applied to preparing medicine for treating wound surfaces and comprise lysozyme, antibacterial peptides and chaperonins. The antibacterial peptides are connected to C tail ends of the lysozyme, and the chaperonins is connected to N tail ends of the lysozyme. The novel application has the advantages that the lysozyme-antibacterial peptide fusion proteins are pure biological preparations, are anti-inflammation, have sterilization functions, have little wound surface irritation or are free of irritation, endogenous growth factor secretion of tissues can be promoted, accordingly, angiogenesis can be promoted, quick growth of granulation tissues and skins can be promoted, the wound surfaces can be quickly healed, effects of quickly treating the wound surfaces can be realized, and the lysozyme-antibacterial peptide fusion proteins are particularly applicable to quickly healing diabetes ulcers.
Description
Technical field
The invention belongs to biochemical field, be specifically related to the new opplication of lysozyme-antibacterial peptide fusion rotein, the particularly application of lysozyme-antibacterial peptide fusion rotein in the medicine of preparation treatment wound surface.
Background technology
The infringement that wound surface is normal skin (tissue) under the external world causes injury the effect such as the factor (as surgical operation, external force, heat, electric current, chemical substance, low temperature) and body intrinsic factor (as local blood supply obstacle) causes, the normal destruction with skin integrity and a certain amount of normal structure Lost lose, simultaneously, the normal function of skin is impaired, also referred to as wound or wound.Wound surface can be divided into acute wound and chronic wound.It is generally acknowledged that acute wound refers to all wound surface within front 2 week of wound surface formation.Common acute wound has operative incision, skin abrasion, burn, skin donor site.Afterwards because some adverse influence factor such as infection, foreign body etc. cause wound to be obstructed, agglutination partially or completely stops, and makes wound healing time be referred to as chronic wound more than the wound surface in 2 week.Common chronic wound has pressure ulcer (decubital ulcer), lower limb vascular (arterialness/veins) ulcer, diabetic ulcer, other difficult healing wound surface.
In recent years, along with China progresses into aging society ranks, chronic wound patient showed increased.In China, chronic wound patient accounts for the 1.5%-3.0% of Surgical Inpatients, is mainly traumatic infection (67.5%), pressure ulcer (9.2%), venous ulcer (6.5%), diabetic ulcer (4.9%) and other (11.9%).Chronic wound may can not threaten patient vitals immediately, if but obstinate, then have a strong impact on patient and household and other quality of life, even can cause and infect diffusion, cause the complication such as sepsis, thus threat to life.And the more difficult healing of diabetic ulcer in all chronic wounds, become the first cause of non-traumatic amputations operation.
Diabetic ulcer refers to that diabetics causes skin infection due to merging neuropathy and the various pathological changes of peripheral vessel in various degree, the diabrosis of burst sick formation and (or) deep tissue.The ulcer of clinical findings diabetic, is not limited to foot, but 96% occurs in limb end, especially based on foot, is therefore called in the world " diabetic foot ".World Health Organization's cri dernier cri disease learns survey result display, and 6.4% of world population in 2010 suffers from diabetes, relates to 2.85 hundred million people, estimates rise to 7.7% to the year two thousand thirty onset diabetes rate, relates to 4.39 hundred million people.In diabetics, there is the patient of 15% ~ 20%, in its course of disease, ulcer of foot or gangrene can occur.And the general more difficult healing of diabetic foot, easily worsen being developed to amputation, and after amputation 5 years survival rates lower than breast carcinoma and carcinoma of prostate.So should to adopt an effective measure in time treatment to chronic wounds such as diabetic foots.
At present, for the formation mechenism of the chronic wounds such as diabetic foot, primary treatments is local anti-infective, promotes about the secretion of Endogenous Growth Factors is to promote angiogenesis, to promote the reparation of defective tissue simultaneously.Existing methods of surgical mainly contains hyperbaric oxygen, local laser illumination, Magnetic heating, autologous stem cell transplantation, application platelet derived growth factor, application Chinese medicine for external application etc.Carry out the internal medicine Primary Care of system simultaneously, comprising: 1. for diabetic ulcer, strict glycemic control; 2. infection control; 3. improve microcirculation, improve nutrition of whole body situation and improve function of nervous system; 4. symptomatic treatment, correction acidosis etc.
And existing patent also discloses the pharmaceutical preparation by utilizing somatomedin to promote skin histology healing.As Chinese patent CN1426813A discloses recombination human epidermal growth factor spray and preparation method; Chinese patent CN101053657A discloses the external preparation for the treatment of intractable cutaneous ulcer, wherein containing epidermal growth factor, antibacterials and insulin; The effective ingredient of the U.S. marketed drug Regranex be the growth of recombination human platelet source because of.
Although these methods above-mentioned and medicine have certain curative effect, all can not meet infection and the requirement promoting the secretion of relevant Endogenous Growth Factors, all very limited target not reaching quickly-healing of effect simultaneously.
Summary of the invention
The object of the invention is the shortcoming for prior art, provide the application of lysozyme-antibacterial peptide fusion rotein in the medicine of preparation treatment wound surface.
First the present invention detects the bactericidal effect of lysozyme-antibacterial peptide fusion rotein by nertralizer qualification test and sterilization experiment.Result show nertralizer solution used can effectively in and lysozyme-antibacterial peptide fusion rotein, and nertralizer solution and neutralized reaction product solution on the growth of staphylococcus aureus substantially without affecting.Under 20 DEG C ± 1 DEG C condition, lysozyme-antibacterial peptide fusion rotein effect 5.0min, to the average kill oncomelania > 5.00 of staphylococcus aureus, meets the requirement of disinfection technology standard.Show that lysozyme-antibacterial peptide fusion rotein has good bactericidal effect.
In a detailed description of the invention, the present invention passes through cell scratch detection lysozyme-antibacterial peptide fusion rotein to the effect of migration of vascular endothelial cells.Result display lysozyme-antibacterial peptide fusion rotein processed group vascular endothelial cell than saline control processed group to periphery move fast.Show that lysozyme-antibacterial peptide fusion rotein has the effect stimulating migration of vascular endothelial cells.
Because diabetic ulcer is the most difficult healing in all wound surface, therefore in a detailed description of the invention, the present invention selects diabetes rat animal model, investigates lysozyme-antibacterial peptide fusion rotein to the impact of wound healing.Result display lysozyme-antibacterial peptide fusion rotein has the effect promoting Wound Healing of Diabetic Rats, can promote the new life of blood capillary in granuloma, can promote that wound tissue secretes the expression of endogenic cell growth factor EGF.
In another embodiment, the present invention investigates the impact of lysozyme-antibacterial peptide fusion rotein on patient with diabetic feet wound healing.After result shows 16 routine patient with diabetic feet use lysozyme-antibacterial peptide fusion rotein liquid, case obvious effective rate 100%.After patient uses lysozyme-antibacterial peptide fusion rotein liquid to treat 10d continuously, wound surface obviously reduces, and fistula road heals, and wound surface is without infection; After treatment 15d, wound surface obviously reduces healing, and wound surface is without infection, and patient discharge, is in voluntarily and uses lysozyme-antibacterial peptide fusion rotein; Follow up a case by regular visits to after treatment 28d, wound surface is fully recovered.And using patient not occur untoward reaction, toleration is good.
Therefore the invention provides the application of lysozyme-antibacterial peptide fusion rotein in the medicine of preparation treatment wound surface.
Wherein, described lysozyme-antibacterial peptide fusion rotein is made up of lysozyme, antibacterial peptide and chaperone, and described antibacterial peptide is connected to the C-terminal of lysozyme, and described chaperone is connected to the N-terminal of lysozyme.Lysozyme is hen egg-white lysozyme (being called for short LYS), and antibacterial peptide (being called for short ABP) is the active fragment of a kind of antibacterial peptide taking from fruit bat.
In some embodiments, chaperone described in described lysozyme-antibacterial peptide fusion rotein is maltose-binding protein (MBP).
In some preferred embodiments, described lysozyme-antibacterial peptide fusion rotein aminoacid sequence is as shown in SEQIDNo:1.
Lysozyme-antibacterial peptide fusion rotein of the present invention is connected with expression vector orientation by technique for gene engineering with the genetic fragment of chaperone by lysozyme, antibacterial peptide, and expression after transformed host cell, extraction, purification obtain.
Wound surface of the present invention can be acute wound or chronic wound.
Wherein, described acute wound is operative incision, skin abrasion, burn, skin donor site.Described chronic wound is pressure ulcer, lower limb vascular ulcer or diabetic ulcer.
Present invention also offers a kind of pharmaceutical preparation for the treatment of wound surface, be made up of the lysozyme-antibacterial peptide fusion rotein of effective dose and pharmaceutically acceptable adjuvant.
The content of the lysozyme-antibacterial peptide fusion rotein described in wherein said pharmaceutical preparation is 10 μ g/mL ~ 100 μ g/mL.
In some preferred embodiments, the content of the lysozyme-antibacterial peptide fusion rotein described in described pharmaceutical preparation is 50 μ g/mL.
Described lysozyme-antibacterial peptide fusion rotein can directly or indirectly be added pharmaceutically acceptable various conventional adjuvants required when preparing different dosage form by those skilled in the art, with traditional drug formulations method, makes conventional external preparation, by applicable mode administration.
It will be understood by those skilled in the art that described external preparation can be solid preparation, as powder or membrane; Semi-solid preparation, as gel, ointment or unguentum; Gaseous formulation, as aerosol; Liquid preparation, as spray or lotion.
In some embodiments, described pharmaceutical preparation is spray.Described spray take pH value as the phosphate buffer of 7.0 is solvent, dissolves lysozyme-antibacterial peptide fusion rotein (MBP-LYS-ABP) and obtains.The content of wherein said lysozyme-antibacterial peptide fusion rotein is 10 μ g/mL ~ 100 μ g/mL.
In some embodiments, described pharmaceutical preparation is gel.Described gel mixes obtained by lysozyme-antibacterial peptide fusion rotein (MBP – LYS-ABP) and substrate.Described substrate can be at least one in hyaluronate sodium, carbomer, carboxymethyl cellulose.
Using dosage and the using method of the pharmaceutical preparation for the treatment of wound surface of the present invention depend on factors, comprise the subjective judgment of the age of patient, body weight, sex, natural health situation, nutriture, administration time, metabolic rate, the course of disease order of severity and diagnosis and treatment doctor.Those skilled in the art can easily determine using dosage and using method according to above-mentioned factor.
As shown from the above technical solution, the invention provides the application of lysozyme-antibacterial peptide fusion rotein in the medicine of preparation treatment wound surface, described lysozyme-antibacterial peptide fusion rotein is made up of lysozyme, antibacterial peptide and chaperone, described antibacterial peptide is connected to the C-terminal of lysozyme, and described chaperone is connected to the N-terminal of lysozyme.Lysozyme-antibacterial peptide fusion rotein is pure biological preparation, antiinflammatory sterilizes, the little or nonirritant to wound surface zest, the endogenic somatomedin of tissue secretion can be promoted simultaneously, thus promotion angiogenesis, promote that granulation tissue and skin grow fast, make wound surface quickly-healing, there is the effect of fast treating wound surface, be particularly useful for the quickly-healing of diabetic ulcer.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 shows embodiment 3 cell scratch experiment result, and wherein scheming A-C is lysozyme-antibacterial peptide fusion rotein processed group, and figure D-F is saline control processed group, and figure A and D is process 0h, figure B and E is process 24h, schemes C and F for process 48h;
Fig. 2 shows the result figure of embodiment 4 5d wound site granuloma Microscopic observation and hair cell vascular counts, and wherein scheming A is lysozyme-antibacterial peptide fusion rotein treatment group H.E coloration result figure, and amplification is 200 times; Figure B is matched group H.E coloration result figure, and amplification is 200 times; Figure C is capillary vascular counts result in matched group and lysozyme-antibacterial peptide fusion rotein treatment group 5d granuloma, wherein bar diagram 1 is matched group capillary vessel number, bar diagram 2 is lysozyme-antibacterial peptide fusion rotein treatment group capillary vessel number, and two groups of data have notable statistics difference (p<0.01);
Fig. 3 shows embodiment 4 5d rat back wound cardinal principle and pathological study result figure, wherein schemes the gross findings figure that A1 is matched group; Figure A2 is the gross findings figure of fusion rotein treatment group; Figure B1 is the H.E result figure of matched group, and amplification is 100 times; Figure B2 is the H.E coloration result figure of fusion rotein treatment group, and amplification is 100 times;
Fig. 4 shows the expressed fusion protein of embodiment 4 lysozyme-antibacterial peptide fusion rotein treatment different time wound tissue EGF, and wherein swimming lane is from left to right followed successively by treatment 1d, 7d, 14d;
Fig. 5 shows the design sketch of embodiment 5 lysozyme-antibacterial peptide fusion rotein treatment diabetic foot, and wherein scheme A for wound surface before treatment, figure B is wound surface after operation wound clearing, and figure C is treatment 10d wound surface; Figure D is treatment 15d wound surface; Figure E is that 28d follows up a case by regular visits to wound surface.
Detailed description of the invention
The invention discloses the application of a kind of lysozyme-antibacterial peptide fusion rotein (MBP-LYS-ABP) in the medicine of preparation treatment wound surface, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Product of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope product as herein described is changed or suitably change with combination, realize and apply the technology of the present invention.
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: the preparation of lysozyme-antibacterial peptide fusion rotein MBP-LYS-ABP
1, the structure of recombiant plasmid pMAL-p2x/LYS-ABP and conversion
Design the peptide sequence of LYS-ABP gene according to Genbank, then design LYS-ABP gene order according to password principle of optimality, by full genome synthesis LYS-ABP antigen-4 fusion protein gene sequence, and add EcoR I-HindIII restriction enzyme site respectively at two ends.After the LYS-ABP fusion protein gene fraction that full genome synthesizes being carried out EcoRI-HindIII is two and cutting, as being inserted into fragment after reclaiming.
Expression vector pMAL-p2x, purchased from NEB company, cuts rear recovery large fragment as carrier using two for pMAL-p2x plasmid EcoRI-HindIII, is connected, then uses CaCl with above-mentioned LYS-ABP fragment T4 ligase
2method is transformed in E.ColiDH5a, and concrete steps are undertaken by the method on " molecular cloning " handbook.Use enzyme action screening positive clone, about the 490bp band person that has an appointment is the plasmid inserting genes of interest, called after pMAL-p2x/LYS-ABP.
The recombinant plasmid transformed filtered out, in expression bacterium BL21 (DE3) pLysS, obtains recombinant expressed bacterium pMAL-p2x-LYS-ABP/BL21 (DE3) pLysS.
2, the expression of target protein
Material: recombinant expressed bacterium pMAL-p2x-LYS-ABP/BL21 (DE3) pLysS; LB culture medium (peptone 10g/L, yeast powder 5g/L, NaCl10g/LpH7.2) is distributed into 250mL, 2000mL triangle shaking flask, 121 DEG C of autoclaving 15min, for subsequent use.
Method: have on the LB flat board of recombinant expressed bacterium pMAL-p2x-LYS-ABP/BL21 (DE3) pLysS bacterium colony long, picking list colony inoculation is in 5mLLB culture medium, 37 DEG C when being cultured to OD600=0.6, in 250mLLB culture medium of transferring, about 15 little the stoppings when OD600=0.6 of amplification culture are cultivated.Be transferred in the 2000mL conical flask containing LB by the recombinant expressed bacterium that 250mLLB cultivates, 37 DEG C, 200rpm, continues to cultivate 20hr, when being cultured to OD600=0.6, adds IPTG to final concentration 1mM in 37 DEG C of inducing culture 3hr.
3, the separation and purification of target protein
The bacterium liquid of abduction delivering is placed in 4 degree of refrigerators, place and make thalline natural sedimentation in 24 hours, after thalline natural sedimentation, remove supernatant, add the buffer (50mMTris-HCl of equivalent, 2mMEDTA, 0.1%TritonX-100, PH8.0), forward in other container, put into-20 degree refrigerators, treat completely freezing after, take out after dissolving completely, then repeat freeze thawing twice.
In the liquid after freeze thawing, add DNA enzymatic with the DNA of thalline release of degrading, room temperature places about 5 hours DNA digestions.
Affinitive layer purification: chromatography media: Amylose-Resin; Chromatographic column specification: internal diameter 50cm, high 40cm, medium height about 10cm, medium volume is about 200mL; Monitoring: 280nm; Balance liquid is 25mMpH8.0Tris-HCl buffer.First with more than balance liquid balance chromatographic column 5 column volumes, flow velocity 20mL/min, then after the bacterium liquid sample balance liquid of DNA digestion being diluted 3 times, carry out loading, flow velocity is 20mL/min, balance about 5 column volumes are continued after having gone up sample, use 25mMpH8.0Tris-HCl buffer, 0.15MNaCl washes foreign protein and is returned to baseline to uv absorption, use eluent (25mMpH8.0Tris-HCl buffer, 0.15MNaCl and 10mM maltose) to carry out eluting, and collection obtain destination protein.Finally regenerate medium with water, the ethanol that medium can add 20% is afterwards put into 4 DEG C and is preserved.Collection obtains destination protein aminoacid sequence as shown in SEQIDNo:1.
The bactericidal effect laboratory research of embodiment 2, lysozyme-antibacterial peptide fusion rotein
1, experiment material
1.1, test sample: the lysozyme-antibacterial peptide fusion rotein that embodiment 1 is obtained.
1.2, bacterial strain: staphylococcus aureus (ATCC6538), purchased from Military Medical Science Institute's Disinfection examination center.
2, experimental technique
2.1, experimental basis: Ministry of Health of the People's Republic of China's " disinfection technology standard " (2002 editions), 2.1.1.5.6 article and 2.1.1.7.5 article.
2.2, nertralizer qualification test:
Experiment grouping:
1st group: disinfectant solution+bacteria suspension is cultivated, observe disinfectant to test organisms with or without killing or rejection ability;
Whether 2nd group: (disinfectant solution+bacteria suspension)+nertralizer is cultivated, observing the test organisms after being subject to Disinfectant effect after residual disinfectancy agent is neutralized can restoration ecosystem;
3rd group: nertralizer+bacteria suspension is cultivated, whether antibacterially observe nertralizer;
4th group: (disinfectant solution+nertralizer)+bacterium liquid carries out cultivation and observes neutralized reaction product, or whether is not had impact by the residual disinfectancy agent neutralized completely to the growth and breeding of test organisms;
5th group: diluent+bacteria suspension is cultivated, contrast as bacterium number;
6th group: diluent+nertralizer+culture medium is cultivated, as negative control.
The obtained lysozyme-antibacterial peptide fusion rotein pH value of embodiment 1 be 7.0 phosphate buffer be that solvent is configured to disinfectant solution and tests, action time is 1.0min, and test temperature is 20 DEG C ± 1 DEG C, test repetition 3 times.
2.3, bactericidal assay: the obtained lysozyme-antibacterial peptide fusion rotein pH value of embodiment 1 be 7.0 phosphate buffer be that solvent is configured to disinfectant solution and tests, action time is 2.5min, 5.0min and 7.5min, and test temperature is 20 DEG C ± 1 DEG C.Test repetition 3 times.
3, result
3.1, nertralizer qualification test: prove through 3 repeated trials, 1 group of average production clump count is 0cfu/ sheet, 2 groups is 197cfu/ sheet, 3 groups, 4 groups and 5 groups of average colony numbers are respectively 1140000cfu/ sheet, 1120000cfu/ sheet and 1147000cfu/ sheet, between three groups, error rate is 2.47%, and concrete outcome is in table 1.Result show nertralizer used can in and the obtained lysozyme-antibacterial peptide fusion rotein of embodiment 1, and nertralizer and neutralized reaction product on the growth of staphylococcus aureus substantially without affecting.
Table 1 nertralizer identification experiment result
3.2, bactericidal assay: under test temperature 20 DEG C ± 1 DEG C condition, prove through 3 repeated trials, the lysozyme-antibacterial peptide fusion rotein effect 5.0min that embodiment 1 is obtained, to the average kill oncomelania > 5.00 of staphylococcus aureus, concrete outcome is in table 2.
Table 2 disinfectant solution is to the killing effect of staphylococcus aureus
Note: the average bacterium number of positive control and scope: 653300cfu/ sheet (575000cfu/ sheet ~ 7575000cfu/ sheet)
4, conclusion:
Nertralizer solution used can effectively in and the obtained lysozyme-antibacterial peptide fusion rotein of embodiment 1, and nertralizer solution and neutralized reaction product solution on the growth of staphylococcus aureus substantially without affecting.Under 20 DEG C ± 1 DEG C condition, the lysozyme-antibacterial peptide fusion rotein effect 5.0min that embodiment 1 is obtained, to the average kill oncomelania > 5.00 of staphylococcus aureus, meets the requirement of disinfection technology standard, has good bactericidal effect.
Embodiment 3, lysozyme-antibacterial peptide fusion rotein promote Effect disquisition 1, the experiment material of migration of vascular endothelial cells
1.1 cell strains: ECV304 cell strain (purchased from microorganism of military medical sciences academy institute).
1.2 test samples: the lysozyme-antibacterial peptide fusion rotein that embodiment 1 is obtained.
2, experimental technique
By cell by 3 × 10
5in/boring ratio example inoculation 24 orifice plates, with aseptic rifle head, multiple tracks cut is done to the cell that culture plate grows after being cultured to 80% fusion, use PBS cyclic washing, change serum-free medium into, continue to cultivate 12h, process cell in vain with the lysozyme-antibacterial peptide fusion rotein that 50 μ g/mL embodiments 1 obtain, take a picture every 12h basis of microscopic observation, absorption supernatant is for subsequent use, establishes saline control group simultaneously.
3, result
In cell scratch experiment, the obtained lysozyme-antibacterial peptide fusion rotein processed group vascular endothelial cell of visible 50 μ g/mL embodiments 1 than saline control processed group to periphery move fast, result is as Fig. 1.Show that the lysozyme-antibacterial peptide fusion rotein that embodiment 1 obtains has the effect stimulating migration of vascular endothelial cells.
Embodiment 4, lysozyme-antibacterial peptide fusion rotein promote the Effect disquisition of diabetes rat animal model wound healing
1. experiment material
1.1 laboratory animals: male Wistar rat 34, body weight is 180 ± 20g, purchased from Military Medical Science Institute's animal center.
1.2 by test product: the lysozyme-antibacterial peptide fusion rotein that embodiment 1 is obtained.
1.3 main agents: EGF protein antibodies (R & D); Horseradish peroxidase-labeled sheep anti-mouse igg, DAB nitrite ion (Beijing Zhong Shan Bioisystech Co., Ltd), poly-D-lysine (Wuhan doctor's moral engineering company); Streptozocin (Merck company).
1.4 instrument and equipments: card punch (Beijing measuring instruments and cutting tools plant), ordinary optical microscope, inverted microscope (Olympus, Japan); TGL-16G high speed tabletop centrifuge (Shanghai); 2300 type CO
2gas incubator (SheldonManufacturingInc., the U.S.); Clean bench (Beijing semiconductor equipment one factory); PhilipsEM400T type transmission electron microscope (Military Medical Science Institute's Instrumental Analysis center).
2. method
2.1 animal model preparations
Rat feeding is after one week, and fasting 24h, tail vein injection is dissolved in the streptozocin 12mg of 0.1mol/L citrate buffer solution, adopts determination of glucose oxidase blood glucose value after 72h, when more than 11.1mol/L, rat blood sugar value can think that diabetes model is successful.All experimental rat blood glucose values all exceed 11.1mol/L.Be divided into experimental group and matched group at random by blood glucose value and body weight, often organize 17.Shave off back hair after 3% amobarbital sodium anesthetized rat, punch with the card punch of diameter 1.5cm, cut three circular wound surface in back, be deep to subcutaneous.Treatment group with obtained lysozyme-antibacterial peptide fusion rotein (the 50 μ g/mL) Direct spraying of embodiment 1 in wound, once a day, treatment 14 days continuously, matched group equivalent PBS.
2.2 cardinal principle and pathological study
Often organize get 5 in the 0th, 1,3,5,7,10,14d observes wound size, each time point of residue rat puts to death 2, fixing in 10% formalin solution after drawing materials, and conventionally makes paraffin section, and pathological study is carried out in H.E dyeing.Get 10 visuals field from 7d wound site granuloma under microscope, carry out blood capillary counting.
2.3Westernblot detects the expression of Diabetic Rat Wound histiocytokine
With the treatment group wound tissue homogenate of the 1st, 7,14 days, collection organization is in lysate A, and operating process is as follows: wash tissue three times with ice-cold PBS, blots clean residual PBS, adds the lysate A of pre-cooling, poured into wherein by tissue homogenate liquid.Protein quantification adopts BCA-200 protein quantification test kit.20 μ g got by each sample, mix, denatured by boiling 5min, carry out 12%SDS-PAGE protein electrophoresis and transferring film with sample-loading buffer and DTT with 8:10:2 ratio.By film with 5% skim milk close 1h, with mouse-anti EGF antibody 4 DEG C of hybridized overnight.Fully wash away non-binding antibody, add the continuation incubated at room 1h of the horseradish peroxidase-labeled of against murine, develop the color with enhanced chemical fluorescein.
3. result
3.1 gross examination of skeletal muscle
0,1,3,5,7,10,14d observes display, with the treatment group rat wound healing quickening more obvious than matched group of the obtained lysozyme-antibacterial peptide fusion rotein of embodiment 1.
Capillary vascular counts in 3.2 granuloma
Get 10 visuals field from 7d wound site granuloma, counting blood capillary quantity is also added up, and the results are shown in Figure 2.
The lysozyme-antibacterial peptide fusion rotein treatment group obtained by the known embodiment of Fig. 2 result 1 is obviously many compared with the blood capillary number of matched group, and has significant difference.Show that lyase-antibacterial peptide fusion protein can promote the new life of blood capillary in granuloma.
3.2 pathological study
The epithelium at the visible wound surface edge of pathological study result is gradually to wound Center shift, without the accessory structure such as hair follicle, sebaceous gland under new epithelize, lysozyme-antibacterial peptide fusion rotein treatment group comparatively control rats wound perimeter epithelization process occurs fast, and horny layer thicker (Fig. 3).
3.3. lysozyme-antibacterial peptide fusion rotein promotes the expression of Diabetic Rat Wound histiocytokine
The wound tissue of Westernblot result display lysozyme-antibacterial peptide fusion rotein treatment different time (1,7 and 14 day) all has the expression of cell growth factor EGF, and treatment time is longer, and the expression of EGF is higher (Fig. 4).Show that lysozyme-antibacterial peptide fusion rotein can promote that wound tissue secretes endogenic cell growth factor EGF, and in regular hour-dose-effect relationship.
4. conclusion
Lysozyme-antibacterial peptide fusion rotein has the effect promoting Wound Healing of Diabetic Rats, can promote the new life of blood capillary in granuloma, can promote that wound tissue secretes the expression of endogenic cell growth factor EGF.
Embodiment 5, lysozyme-antibacterial peptide fusion rotein promote the Effect disquisition of patient with diabetic feet wound healing
1 materials and methods
1.1 physical data
1.1.1 the diagnostic criteria of diabetic foot
The grade scale formulated by classification and nineteen ninety-five Chinese Medical Association first national diabetic foot Symposium Held of international wagner is divided into V level: O level: acra blood supply insufficiency; I level: skin has open focus; II level: infection focus invades deep muscle tissue; III level: tendon ligament damage; IV level: bony defect; V level: large portion or full foot gangrene, even involves ankle joint and shank.
16 routine patient male 8 examples, women 8 example; The oldest 73 years old, minimum 44 years old; Diabetic history is the longest 30 years, the shortest 2 years, is type 2 diabetes mellitus patient.Diagnostic classification case: I level 4 example, II level 4 example, III level 3 example, IV level 3 example, V level 2 example.
1.2 Therapeutic Method
16 routine patients all use insulin strictly to control outside protopathy blood glucose, the person that has focus of infection, according to the cultivation of cervical secretions of local necrosis, select responsive and enough broad ectrum antibiotic.
Topical therapeutic: conventional debridement, need patients with amputation amputation, then on wound surface, directly spray the obtained lysozyme-antibacterial peptide fusion rotein liquid of embodiment 1, rear sterile gauze covers, and changes dressings every day once, the course for the treatment of surrounding, Estimating curative effect at the end of surrounding.
1.3 curative effect determinate standard
Clinical cure: ulcer wound surface heals completely, recovers walking function;
Effective: ulcer wound surface reduces or wound granulation rate of growth more than 60%;
Effective: ulcer wound surface reduces or wound granulation rate of growth more than 30%;
Invalid: ulcer does not heal, wound surface develops, row amputation or the died because of complication.
2 results
2.1 patient outcomes
After the lysozyme-antibacterial peptide fusion rotein liquid using embodiment 1 obtained, case obvious effective rate 100%.Model case is shown in Fig. 5.After patient uses the obtained lysozyme-antibacterial peptide fusion rotein liquid of embodiment 1 to treat 10d continuously, wound surface obviously reduces, and fistula road heals, and wound surface is without infection; After treatment 15d, wound surface obviously reduces healing, and wound surface is without infection, and patient discharge, is in voluntarily and uses lysozyme-antibacterial peptide fusion rotein; Follow up a case by regular visits to after treatment 28d, wound surface is fully recovered.
2.2 adverse drug effects
After the lysozyme-antibacterial peptide fusion rotein using embodiment 1 obtained, there is not untoward reaction in patient, toleration is good.
Embodiment 6, lysozyme-antibacterial peptide fusion rotein spray
Take pH value as the phosphate buffer of 7.0 be solvent, dissolve the lysozyme-antibacterial peptide fusion rotein that embodiment 1 is obtained.The content of wherein said lysozyme-antibacterial peptide fusion rotein is 50 μ g/mL.
Embodiment 7, lysozyme-antibacterial peptide fusion rotein gel.
The lysozyme-antibacterial peptide fusion rotein that embodiment 1 is obtained and hyaluronate sodium mixing obtain, and the content of wherein said lysozyme-antibacterial peptide fusion rotein is 50 μ g/mL, and the content of hyaluronate sodium is 0.1% (mass fraction).
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. the application of lysozyme-antibacterial peptide fusion rotein in the medicine of preparation treatment wound surface, described lysozyme-antibacterial peptide fusion rotein is made up of lysozyme, antibacterial peptide and chaperone, described antibacterial peptide is connected to the C-terminal of lysozyme, and described chaperone is connected to the N-terminal of lysozyme.
2. application according to claim 1, described in described lysozyme-antibacterial peptide fusion rotein, chaperone is maltose-binding protein, and described lysozyme-antibacterial peptide fusion rotein aminoacid sequence is as shown in SEQIDNo:1.
3. application according to claim 1 and 2, described wound surface is chronic wound.
4. application according to claim 3, described chronic wound is pressure ulcer, lower limb vascular ulcer or diabetic ulcer.
5. treat a pharmaceutical preparation for wound surface, be made up of the lysozyme-antibacterial peptide fusion rotein of effective dose and pharmaceutically acceptable adjuvant.
6. pharmaceutical preparation according to claim 5, the content of described lysozyme-antibacterial peptide fusion rotein is 10 μ g/mL ~ 100 μ g/mL.
7. pharmaceutical preparation according to claim 5, described pharmaceutical preparation is external preparation.
8. pharmaceutical preparation according to claim 7, described external preparation is solid preparation, semi-solid preparation, gaseous formulation or liquid preparation.
9. pharmaceutical preparation according to claim 8, described solid preparation is powder or membrane; Described semi-solid preparation is gel, ointment or unguentum; Described gaseous formulation is aerosol; Described liquid preparation is spray or lotion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510478272.7A CN105169377A (en) | 2015-08-06 | 2015-08-06 | Novel application of lysozyme-antibacterial peptide fusion proteins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510478272.7A CN105169377A (en) | 2015-08-06 | 2015-08-06 | Novel application of lysozyme-antibacterial peptide fusion proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105169377A true CN105169377A (en) | 2015-12-23 |
Family
ID=54892124
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510478272.7A Pending CN105169377A (en) | 2015-08-06 | 2015-08-06 | Novel application of lysozyme-antibacterial peptide fusion proteins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105169377A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112111474A (en) * | 2020-09-29 | 2020-12-22 | 青岛红樱桃生物技术有限公司 | Recombinant lysozyme LYZ-2 with improved enzyme activity, and mutant and application thereof |
| CN112353937A (en) * | 2020-12-03 | 2021-02-12 | 周清华 | Fresh agrimony freeze-dried powder composite preparation |
| CN114213551A (en) * | 2021-12-03 | 2022-03-22 | 四川农业大学 | High-expression recombinant biological protein API and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649311A (en) * | 2009-09-15 | 2010-02-17 | 吉林大学 | Preparation method of human lysozyme-antibacterial peptide Catesbeianin-1 fusion protein and application of same on preventing and curing cow mastitis |
| CN104761621A (en) * | 2014-01-06 | 2015-07-08 | 长春新悦然科技有限公司 | Antibacterial proteins |
-
2015
- 2015-08-06 CN CN201510478272.7A patent/CN105169377A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649311A (en) * | 2009-09-15 | 2010-02-17 | 吉林大学 | Preparation method of human lysozyme-antibacterial peptide Catesbeianin-1 fusion protein and application of same on preventing and curing cow mastitis |
| CN104761621A (en) * | 2014-01-06 | 2015-07-08 | 长春新悦然科技有限公司 | Antibacterial proteins |
Non-Patent Citations (5)
| Title |
|---|
| YIGONG GE等: "In Vitro Antibacterial Properties of Pexiganan, an Analog of Magainin", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| 侯国宾 等: "抗菌肽临床应用前景分析", 《生命科学》 * |
| 史春林 等: "抗菌肽在宿主防御中的作用", 《中国生物工程杂志》 * |
| 马青山 等: "抗菌肽融合表达研究进展", 《生物工程学报》 * |
| 黎观红 等: "抗菌肽的抗菌作用及其机制", 《动物营养学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112111474A (en) * | 2020-09-29 | 2020-12-22 | 青岛红樱桃生物技术有限公司 | Recombinant lysozyme LYZ-2 with improved enzyme activity, and mutant and application thereof |
| CN112111474B (en) * | 2020-09-29 | 2022-08-30 | 青岛红樱桃生物技术有限公司 | Recombinant lysozyme LYZ-2 with improved enzyme activity, and mutant and application thereof |
| CN112353937A (en) * | 2020-12-03 | 2021-02-12 | 周清华 | Fresh agrimony freeze-dried powder composite preparation |
| CN114213551A (en) * | 2021-12-03 | 2022-03-22 | 四川农业大学 | High-expression recombinant biological protein API and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103665111B (en) | Variant HC-15 of a kind of chittue antibacterial peptide Hc-CATH and its preparation method and application | |
| CN101412753A (en) | Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof | |
| CN105169377A (en) | Novel application of lysozyme-antibacterial peptide fusion proteins | |
| US20210038690A1 (en) | Promoting epithelial regeneration using heparin binding epidermal growth factor like growth factor | |
| CN110642924B (en) | Skin protection peptide OM-TV16 and preparation method and application thereof | |
| EP2450440A1 (en) | Fibrinogenolytic enzyme tabfiblysin of horsefly, tabanus yao, encoding gene and use thereof | |
| CN107308441A (en) | Stem cell culture supernatant gel combination and its preparation method and application | |
| CN111423516A (en) | Protein and application thereof in wound repair and bacteriostasis | |
| Navadiya et al. | Study of topical placental extract versus povidone iodine and saline dressing in various diabetic wounds | |
| CN114716515A (en) | Polypeptide analogue and preparation method and application thereof | |
| KR20150108351A (en) | Protein slurp-1 for use in the treatment of ocular diseases | |
| CN100334114C (en) | Novel fusion protein production and uses | |
| CN114601912A (en) | Itch-caused polypeptide caused by tick and mosquito bites and itch-resisting application thereof | |
| CN102924607B (en) | OGP-AcAP5 fusion protein for treating osteoporosis and thrombosis and nucleic acid for coding same | |
| CN102816206B (en) | Synthetic peptide and application thereof | |
| CN103031268B (en) | OGP-rhLeptin fusion protein transgenic engineering strain | |
| CN101721681A (en) | Application of human Keratiocyte growth factor 1 in preparation of medicament for treating foot ulcers and leg ulcers of diabetes | |
| CN104356219B (en) | A kind of Xanthopsyllacheopis antiarrhythmic polypeptide and preparation method thereof | |
| CN103007260A (en) | Preparation method of medicine for rapidly healing burned tissue | |
| CN107158361B (en) | Application of the REG1A albumen in preparation treatment and/or prevention retinal cell apoptosis drug | |
| CN102964452B (en) | For the msCT-rhLeptin fusion rotein of curing osteoporosis with obesity and the nucleic acid of this fusion rotein of encoding | |
| CN103012598B (en) | OGP-rhLeptin fusion protein for treating osteoporosis and obesity and nucleic acid encoding same | |
| Das et al. | Animal-Derived products as Medicinal Products | |
| CN112675294A (en) | Preparation of fibroblast and application of fibroblast in plastic surgery repair | |
| CN102911907B (en) | OGP-AcAP5 (Osteogenic Growth Peptide-Ancylostoma Caninum Anticoagulant Peptide 5) fusion protein transgenic engineering strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151223 |
|
| RJ01 | Rejection of invention patent application after publication |